Figure 5.

Duplex RNAs were not detected in HeLa cells using RPA. (a) Ribonuclease protection assay (RPA). of cytoplasmic RNA. Lane 1, HeLa lysate -RNAse; lane 2, HeLa lysate +RNAse; lane 3, HeLa overexpressing sense (S) -RNAse; lane 4, HeLa overexpressing sense (S) +RNAse; lane 5, HeLa overexpressing antisense (AS) -RNAse; lane 6, HeLa overexpressing antisense (AS) +RNAse; lane 7, HeLa overexpressing hairpin vector (S-AS) -RNAse; lane 8, HeLa overexpressing hairpin vector (SAS) +RNAse; lane 9, HeLa transfected with in vitro transcribed S-AS RNA duplex -RNAse; lane 10, HeLa transfected with in vitro transcribed S-AS RNA duplex +RNAse. All of the +RNAse samples treated with RNAse A+T, along with -RNase samples, were separated on denaturing PAGE and probed for the overlap region of TS mRNA. The predicted positive bands (rTSα, 1,800 bp endogenous antisense mRNA; TS 1,600 bp endogenous sense mRNA and 1,100 bp vector based S-AS mRNA) were detected in RNAse negative samples and revealed efficacy of RNAse treatment as well as specificity of the probe. Additionally, signals corresponding to a 200 bp product (protected overlap region) were seen only in the last four lanes, which had synthetically endogenous or exogenous RNA duplex. (b) Additional controls for RPA of cytoplasmic RNA. Lane 1, cytoplasmic lysate of HeLa cells; lane 2, cytoplasmic lysate of HeLa cells overexpressed with sense and antisense vector; lane 3, lysate from HeLa cells transfected with in vitro transcribed S-AS RNA duplex; and lane 4, total RNA from HeLa cells overexpressing sense and antisense vector. All samples were treated with RNAse A+T, separated on denaturing PAGE and probed for overlapping region of TS mRNA. The expected 200 bp product (protected overlapping region) was seen only in lane 3, which included exogenous synthetic RNA duplex.